The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence which codes for a haemocyanin, a haemocyanin domain or a fragment with the immunological properties of at least one domain of haemocyanin, and comprising at least one intron sequence, constructs which comprise such molecules, host cells which comprise the nucleic acid sequences or the constructs, processes for the preparation of haemocyanin polypeptides, and recombinant haemocyanin polypeptides.
Haemocyanin is a blue copper protein which occurs in a freely dissolved form in the blood of numerous molluscs and arthropods and transports oxygen. Of the molluscs, the cephalopods, chitons, most gastropods and some bivalves contain haemocyanin. Among the arthropods, haemocyanin is typical of arachnids, xiphosurans, malacostracan crustaceans and Scutigera. Numerous species of insects contain proteins which are derived from haemocyanin. Haemocyanins are present in the extracellular medium and float in the haemolymph.
While arthropod haemocyanin has a maximum diameter of 25 nm under an electron microscope and a subunit has a molecular weight of 75,000 Da, mollusc cyanins are much larger. Thus e.g. the haemocyanin of Megathura has a diameter of 35 nm and is composed of 2 subunits. Each subunit has a molecular weight of approx. 400,000 Da and is divided into eight oxygen-binding domains, each of which has a molecular weight of approx. 50,000. The domains differ immunologically. These domains can be liberated from the subunit by limited proteolysis.
The haemocyanin of gastropods visible under an electron microscope has a molecular weight of approx. 8 million Da and is a di-decamer. In contrast to this, the haemocyanin of cephalopods is arranged as an isolated decamer, which also differs significantly from the haemocyanin of gastropods in the quaternary structure.
The haemocyanin of the Californian keyhole limpet Megathura crenulata is of particular immunological interest. The haemocyanin is therefore also called keyhole limpet haemocyanin (KLH). Haemocyanins are very potent antigens. Immunization of a vertebrate leads to a non-specific activation of the immune system which to date is not very well understood. By the general activation of the immune system, it is then possible also to achieve an immune reaction to other foreign structures which have previously been tolerated. KLH is used above all as a hapten carrier in order thus to achieve the formation of antibodies against the hapten.
In addition to Megathura crenulata, the abalone Haliotis tuberculata also belongs to the Archaegastropoda group, which is relatively old in respect of evolution. It is known that Haliotis also produces haemocyanin.
KLH is a mixture of two different haemocyanins, which are called KLH1 and KLH2. The subunit of KLH1 is a 390 kDa polypeptide which consists of eight globular domains called 1 a to 1 h according to their sequence in the subunit. On the other hand, KLH2 has a molecular weight of 350 kDa and according to the most recent data also contains 8 domains, called 2 a to 2 h. In vivo every type of subunit forms homo-oligomers, while no hetero-oligomers have been observed.
Amino-terminal, internal and carboxy-terminal domains have been obtained by limited proteolysis and crossed immunoelectrophoresis of the subunit of KLH1 and KLH2, and their amino-terminal sequences has been determined (Söhngen et al., Eur. J. Biochem. 248 (1997), 602–614; Gebauer et al., Zoology 98(1994), 51–68). However, the resulting sequences do not allow designing of sequence-specific primers and/or probes which promise success for hybridization with genomic DNA. Although both KLH types have been known since 1991 and 1994 respectively, it has so far not been possible to clarify the primary structure.
At the DNA level, in respect of molluscs only the cDNA sequence of the haemocyanin subunit from the cephalopod Octopus dofleini is so far known (Miller et al., J. Mol. Biol. 278 (1998), 827–842). Octopus dofleini is phylogenetically very far removed from the archaegastropods. A haemocyanin gene sequence from molluscs is so far not known at all.
As described by Miller at al. supra, it is difficult both to isolate a single functional domain (functional unit=domain; also called functional domain) and to obtain tissue which is suitable for purification of mRNA for cDNA sequencing.
There is a further difficulty in the analysis of the haemocyanin from Megathura crenulata in that the test animals must have reached an age of 4 to 8 years for haemolymph to be taken from them in the first place. After the haemolymph has been taken, haemocyanin is not subsequently produced in these animals. It is not yet known how haemocyanin synthesis could be stimulated. Furthermore, culture of Megathura is extremely expensive, since special flow basins are required for this.